PROJECT SUMMARY Neurodevelopmental disorders (NDDs), including syndromes of intellectual disability (ID) are early-presenting cognitive disorders that affect 1-8% of the population. Recent genome-wide studies that have sought the genetic basis of ID have implicated genes responsible for synaptic function, transcriptional regulation, and enzymes that modulate the post-translational methylation of histones, proteins around which DNA is wrapped. This proposal seeks to address the gap in knowledge around why disruption of histone methylation dynamics frequently leads to cognitive deficits by investigating brain-specific alternative splicing of histone-regulating genes. Recent work has shown that the histone demethylase, LSD1, has a neuronal isoform leading to altered substrate specificity. PHF21A is a histone reader that acts in complex with LSD1 such that it canonically recognizes the product of the LSD1 reaction. Loss of function of LSD1 and PHF21A lead to ID syndromes, Kabuki Syndrome and Potocki Shaffer Syndrome (PSS), respectively. My preliminary data shows that PHF21A also has a neuronal-specific isoform (PHF21A-n). Through an RNA-Seq study of PSS patients, I also found that loss of PHF21A function leads to transcriptional downregulation of signaling pathways important for learning and memory. Our lab previously generated a Phf21a homozygous null mouse that died as a result of an inability to suckle milk. No structural brain abnormalities or neuron morphological abnormalities, but synaptic formation, a phenotype relevant to NDDs, was not assessed. Given the literature and my preliminary data, my central hypothesis is that PHF21A-n has unique histone binding properties that allow for proper synaptic formation in maturing neurons. In this proposal, I will test this hypothesis by (1) biochemical analysis of PHF21A-n function by performing binding, transcriptional reporter, and demethylation assays, in combination with canonical and neuronal isoforms of LSD1. I will next (2) assess the contributions of each PHF21A isoform in synaptic development using a neuron culture system with Phf21a null cultured neurons with each isoform individually replaced by transfection. I will evaluate changes in synaptic development immunohistochemically and identify programs of PHF21A isoform-specific transcriptional regulation using RNA-Seq. Completion of this work will benefit our understanding of the underlying mechanisms of NDDs given that this proposal aims to study several pathways known to be affected in NDDs: synaptic formation, epigenetic regulation, and alternative splicing. Additionally, completion of this work will provide me with the scientific, technical, and medical training, as detailed in my training plan, which will propel me into a successful career as a physician scientist studying the epigenetic basis of pediatric neurological disease.